Activation of NF-κB regulates numerous cellular processes including cytokine and adhesion molecule expression, cell-cycle regulation and cell growth. Constitutive or excessive NF-κB activity is thus observed in inflammatory diseases, autoimmunity and cancer. Notably, active NF-κB also protects against apoptosis which may contribute to the accumulation of inflammatory, self-reactive or cancerous cells.
Extensive research has been conducted throughout the last decade towards understanding the molecular mechanism, signaling pathways and physiological role of proteins containing the death domain fold (DDF) associated with NF-κB signaling and apoptosis induction. Four related protein-protein interaction domains comprise the DDF superfamily: the death domain (DD); the death effector domain (DED); the caspase recruitment domain (CARD) and the pyrin domain (PYD). These domains share a similar tertiary structure, a 5 to 6 α-helical bundle that likely function as homotypic interaction modules. Acting as signaling adapters and recruiters of proteins into sub-cellular complexes domains of this type participate in the promotion and regulation of apoptosis, innate immunity and cancer development.
Pyrin motifs are generally found at the N-terminus of multidomain proteins. Pyrin (marenostrin), the founding member of the family, is associated with Familial Mediterranean Fever (FMF), an autosomal recessive disease characterized by sporadic attacks of fever and intense abdominal, joint and chest pain. Following its PYD, Pyrin contains a B-box zinc finger and a SPRY domain believed to function as adaptor and ligand binding units, respectively. Interferon inducible (IFI) genes, which code for the HIN-200 family of hematopoetic nuclear proteins and contain one or two copies of a 200-amino-acid domain believed to mediate protein/protein interactions, are preceded by a PYD. Evidence supporting the role of HIN-200 proteins in controlling cell proliferation and differentiation has been previously documented. The pyrin domain of Apoptosis speck protein containing a CARD, ASC (TMS1/Pycard) is followed by a CARD domain. ASC associates with PYD- or CARD-containing proteins through homotypic interactions via both domains. In proapoptotic cells, ASC assembles in large multimeric perinuclear complexes or ‘specks’ by interacting with PYD-containing proteins. The physiological significance of these interactions and that of the speck structure itself remains unclear. ASC also participates in the formation of the inflammasome complex that processes pro IL-1β into the mature form and is up-regulated in neutrophils during inflammation. Two thirds of the 21 members of the recently discovered CATERPILLER (CLR) gene family thought to play an important role in vertebrate immunity functioning as intracellular pathogen-recognizing units, encode a PYD followed by a nucleotide binding domain (NBD) and leucine-rich repeats domain (LRR). CLR proteins have been postulated as modulators of ongoing inflammatory responses and as inducers of apoptosis. Some PYD-containing proteins of the CLR family have been reported to induce NF-κB activation in conjunction with ASC, including PYPAF5, Monarch, and CIAS1. Not surprisingly, mutations in CIAS1/NALP3 associated with the inflammatory syndromes familial cold urticaria, Muckle-Wells, and chronic infantile neurologic cutaneous articular syndrome, showed increased capacity for ASC-dependent NF-κB activation. Other CLRs, such as PAN2/PYPAF4 mediate NF-κB suppression. Although the NBD/LRR of CIAS1 inhibits NF-κB nuclear import, the pyrin domain of PAN2/PYPAF4 inhibits NF-κB, a function that may be mediated through the IKK complex.
The NF-κB family of transcription factors is comprised of five members in mammals: p65 (Rel A), Rel B, c-Rel, p50/p105 and p52/p100, existing as homo- or heterodimers bound to the IκB inhibitory complex in the cytosol. Upon induction by proinflammatory stimuli such as TNF-α or LPS, IκBα is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated and degraded by the 26S proteasome. This process unmasks NF-κB's nuclear localization sequence, leading to its translocation into the nucleus. Binding of NF-κB to its cognate DNA response elements induces the transcription of a host of cytokines and growth factors (e.g. IL-2, II-8, IFN-β, M-CSF, G-CSF, VEGF), as well as various transcription factors and signaling regulators (e.g. IκBα, IRF-1, IRF-2). The pivotal role of NF-κB in biological processes modulating the immune response suggests that localization and subsequent activation must be rigorously controlled. Dysregulation of these events leads to aberrant gene expression associated with numerous human diseases including cancer, neurodegenerative disorders, arthritis and chronic inflammation.
Solitary pyrin domains may disrupt pyrin domain interactions, potentially blocking the formation of speck and/or inflammasome complexes and their downstream effects. Genes comprised exclusively of a PYD have recently been identified in the human genome and in the genomes of pox viruses. Pyrin-only protein 1 (POP1), is encoded by a gene in the close proximity to ASC, suggesting that Pop1 might have arisen from an ancient gene duplication event. POP1 is predominantly expressed in immune tissues where it appears to inhibit NF-κB- and surprisingly enhances caspase-1-activation. Other genes encoding Pyrin-only proteins have been found in the genomes of Capripoxviridae, Leporipoxviridae, Suipoxviridae, and Yatapoxviridae. Recently, Johnston et al. reported that M13L-PYD (a Pyrin-only protein) from myxoma virus inhibits both NF-κB activity and Caspase-1-dependent IL-1b production and M13L deletion was sufficient to inhibit virus replication in vivo. (Johnston, J. B., et al. A poxvirus-encoded pyrin domain protein interacts with ASC-1 to inhibit host inflammatory and apoptotic responses to infection. Immunity 23:587). Therefore, PYDs have an active role as suppressors of host immunity. Modulating NF-κB signaling pathways is one potential mechanism for such suppression.
The completion of the human genome and the sequencing of the genomes of other species have resulted in the emergence of novel genes and the identification of new gene families. The recently described CLR family encompasses PYD-proteins with roles in both innate and acquired immunity. Because of our interest in this gene family, we sought to identify new PYD-encoding genes.